Anti-neovasculature preparations for cancer

ABSTRACT

Disclosed herein are immunogenic compositions, methods of designing immunogenic compositions, methods of treatment using immunogenic compositions, methods of evaluating cell-mediated immunity resulting from immunogenic compositions, research models, and methods of making research models, all of which relate to targeting tumor vasculature.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.10/094,699, filed Mar. 7, 2002, entitled “ANTI-NEOVASCULATUREPREPARATIONS FOR CANCER,” which claims priority under 35 U.S.C. § 119(e)to U.S. Provisional Patent Application Ser. No. 60/274,063, filed onMar. 7, 2001, entitled “ANTI-NEOVASCULATURE PREPARATIONS FOR CANCER,”each of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Description of the Related Art

The treatment of cancer has remained challenging despite the advances inbiomedicine. In recent years two approaches have been described showingmuch promise: therapeutic vaccines and anti-angiogenesis.

Therapeutic vaccines rely on the observation that cancerous tissuesgenerally express certain antigens preferentially, collectivelytumor-associated antigens (TuAA). TuAA include proteins normallyexpressed selectively by the tissue from which the cancer derives(differentiation antigens), proteins that are associated with adifferent stage of development (oncofetal and cancer-testis antigens),proteins that are created by aberrant chromosomal rearrangement, orproteins that are derived from oncogenic viruses. These TuAA, orfragments of them, are then used as immunogens in vaccines intended tostimulate cellular immunity, particularly cytotoxic T lymphocytes (CTL),capable of killing the tumor cells.

The anti-angiogenesis approach takes advantage of the need of tumors torecruit a blood supply to support their continued growth. To accomplishthis, tumors secrete angiogenic factors that promote the growth of newblood vessels. The anti-angiogenesis approach aims to disrupt a tumor'ssupply of nutrients to cause it to die, or at least limit its growth.Attempts at this approach have sought chemotherapeutic drugs useddirectly against a variety of anti-angiogenic factors and angiogenesis.

SUMMARY OF THE INVENTION

The invention disclosed herein is directed to compositions designed tostimulate cellular immune responses targeting tumor-associatedneovasculature (TuNV). In one embodiment of the invention thecompositions stimulate a CTL response. Such compositions may include oneor more epitopes of the target antigen. One aspect of this embodimentspecifically includes a housekeeping epitope, another specificallyincludes an immune epitope or epitope cluster, and another aspectspecifically combines housekeeping and immune epitopes.

Embodiments of the invention relate to the use of prostate specificmembrane antigen (PSMA) as the target antigen of the composition.Aspects of this embodiment include various epitopes derived from PSMAprovided directly as polypeptide, or as a nucleic acid capable ofconferring expression of the epitope. Other embodiments relate to theuse of other TuNV-associated antigens.

In other embodiments of the invention, compositions are directed againstboth the TuNV and against TuAA expressed by the cancerous cells, bycombining immunogens derived from both sources into a single formulationor method or treatment.

Preclinical evaluation of the compositions of this invention can beaccomplished using adoptive transfer of immunized T cells into SCID micebearing microvasculature formed from implanted human dermalmicrovascular endothelial cells (HDMEC). Preclinical evaluation can alsobe accomplished through the use of HLA-transgenic mice immunized withcompositions comprised of epitopes conserved between mice and humans.

Embodiments of the invention relate to methods of evaluatingcell-mediated immunity. The methods can include the steps of implantingvascular cells into an immunodeficient mammal; establishing an immuneresponse in the mammal; and assaying a characteristic to determinecell-mediated immunity in the mammal. The cell-mediated immunity can bedirected against a neovasculature antigen, for example. Theneovasculature antigen can be preferentially expressed bytumor-associated neovasculature, for example, and in preferredembodiments can be prostate specific membrane antigen (PSMA), vascularendothelial growth factor receptor 2 (VEGFR2), and the like. Theestablishing step can be achieved, for example, by adoptive transfer ofT-cells to the mammal, by contacting the mammal with an antigen, and thelike. The cell-mediated immunity can be mediated by cytotoxic Tlymphocytes. The vascular cells can be vascular endothelial cells, suchas, for example, human dermal microvascular endothelial cells (HDMEC),telomerase-transformed endothelial cells, and the like. Theimmunodeficient mammal can be a mouse, such as for example a SCID mouse.The characterizing step can include assessing a parameter, such as forexample, vessel formation, vessel destruction, vessel density,proportion of vessels carrying blood of the host mammal, and the like.

The methods can further include the step of implanting tumor cells ortumor tissue in the mouse. The characterizing step can include assessinga parameter, such as, for example, tumor presence, tumor growth, tumorsize, rapidity of tumor appearance, dose of vaccine required to inhibitor prevent tumor establishment, tumor vascularization, a proportion ofnecrotic tissue within the tumor, and the like.

The methods can further include the steps of providing a firstpopulation of mammals and a second populations of mammals; establishingcell-mediated immunity in the first population; differentiallyestablishing cell-mediated immunity in the second population; andcomparing a result obtained from the first population of mammals to aresult obtained from the second population of mammals. The cell-mediatedimmunity of the first population can include, for example, naïveimmunity, immunity to an irrelevant epitope, and the like.

Other embodiments relate to methods of evaluating cell-mediatedimmunity, including immunity directed against a neovasculature antigen.The methods can include the steps of implanting or injectingMHC-transgenic tumor cells into an MHC-transgenic mammal; establishingan immune response in the mammal; and assaying a characteristic todetermine cell-mediated immunity in the mammal. The MHC-transgenicmammal can be an HLA-transgenic mammal, such as, for example an HLA-A2transgenic mammal. In preferred embodiments the mammal can be a mouse.The cell-mediated immunity can be established by vaccination, which inpreferred embodiments can take place prior to, concurrent with, orsubsequent to transfer of the tumor cells, for example. In preferredembodiments the cell-mediated immunity can be mediated by cytotoxic Tlymphocytes. The neovasculature antigen can be preferentially expressedby tumor-associated neovasculature and can also be a tumor-associatedantigen. Preferably, the antigen can be the ED-B domain of fibronectin.The characterizing step can include, for example, assessing a parameter,including tumor presence, tumor growth, tumor size, rapidity of tumorappearance, dose of vaccine required to inhibit or prevent tumorestablishment, tumor vascularization, a proportion of necrotic tissuewithin the tumor, and the like. The methods can further include thesteps of providing a first population of mammals and a secondpopulations of mammals; establishing cell-mediated immunity in the firstpopulation; differentially establishing cell-mediated immunity in thesecond population; and comparing a result obtained from the firstpopulation of mammals to a result obtained from the second population ofmammals. The cell-mediated immunity of the first population can includenaïve immunity, immunity to an irrelevant epitope, and the like.

Still further embodiments relate to methods of treating neoplasticdisease, including the step of immunizing a mammal to induce a cellularimmune response directed against an antigen differentially expressed bytumor-associated neovasculature. The differentially expressed antigencan be a protein, such as, for example prostate specific membraneantigen, vascular endothelial growth factor receptor 2 (VEGFR2), and thelike. In other preferred embodiments, the antigen can be the ED-B domainof fibronectin. The immunization can be carried out, for example, withat least one peptide derived from the sequence of the protein, with anucleic acid capable of conferring expression of the protein orpeptides, and the like. The at least one peptide can include ahousekeeping epitope, for example, and in preferred embodiments can beco-C-terminal with the housekeeping epitope. The methods can furtherinclude at least one additional peptide, wherein the at least oneadditional peptide includes an immune epitope. The methods can includean additional step wherein the mammal is treated with an anti-tumortherapy active directly against cancerous cells. The anti-tumor therapycan be immunization against a tumor-associated antigen. Preferably, thecellular immune response can include a CTL response.

Other embodiments relate to immunogenic compositions. The immunogeniccompositions can include at least one immunogen corresponding to anantigen expressed by tumor-associated neovasculature, wherein thecomposition can induce a cellular immune response. The immunogen can beone that is not associated with a cell conspecific with the recipient.The antigen can be a protein, such as, for example prostate specificmembrane antigen, vascular endothelial growth factor receptor 2(VEGFR2), and the like. In other preferred embodiments the antigen canbe the ED-B domain of fibronectin. The immunogen can include at leastone peptide. The compositions can include a nucleic acid capable ofconferring expression of the antigen, and wherein the antigen is aprotein or a peptide. The compositions can include at least one peptidethat includes a housekeeping epitope, and in preferred embodiments theat least one peptide can be co-C-terminal with the housekeeping epitope.Also, the compositions can additionally include at least one peptidethat includes an immune epitope. The compositions can include at leastone immunogen corresponding to a tumor-associated antigen. In preferredembodiments the cellular immune response can include a CTL response.

Embodiments relate to methods of anti-tumor vaccine design. The methodscan include the steps of identifying an antigen differentially expressedby tumor-associated neovasculature; and incorporating a component of theantigen into a vaccine. The component can include, for example, apolypeptide fragment of the antigen, a nucleic acid encoding the antigenor a fragment of the antigen, and the like.

Further embodiments relate to methods of making a research model. Themethods can include implanting a vascular cell and a tumor cell into animmunodeficient mammal. The tumor cell and the vascular cell can beimplanted adjacent to one another. The vascular cell can be a vascularendothelial cell, such as for example HDMEC. In preferred embodimentsthe vascular endothelial cell can be telomerase-transformed. Theimmunodeficient mammal can be a mouse, such as, for example, a SCIDmouse.

Other embodiments relate to research models. The research models caninclude an immunodeficient mammal. The mammal can include an implantedvascular cell and an implanted tumor cell. The vascular cell and thetumor cell can be implanted adjacent to one another.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, B, and C show results of N-terminal pool sequencing of a T=60min. time point aliquot of the PSMA₁₆₃₋₁₉₂ proteasomal digest.

FIG. 2 shows binding curves for HLA-A2:PSMA₁₆₈₋₁₇₇ andHLA-A2:PSMA₂₈₈₋₂₉₇ with controls.

FIG. 3 shows results of N-terminal pool sequencing of a T=60 min. timepoint aliquot of the PSMA₂₈₁₋₃₁₀ proteasomal digest.

FIG. 4 shows binding curves for HLA-A2:PSMA₄₆₁₋₄₆₉, HLA-A2:PSMA₄₆₀₋₄₆₉,and HLA-A2:PSMA₆₆₃₋₆₇₁, with controls.

FIG. 5 shows the results of a γ(gamma)-IFN-based ELISPOT assay detectingPSMA₄₆₃₋₄₇₁-reactive HLA-A1⁺ CD8⁺ T cells.

FIG. 6 shows blocking of reactivity of the T cells used in FIG. 10 byanti-HLA-A1 mAb, demonstrating HLA-A1-restricted recognition.

FIG. 7 shows a binding curve for HLA-A2:PSMA₆₆₃₋₆₇₁, with controls.

FIG. 8 shows a binding curve for HLA-A2:PSMA₆₆₂₋₆₇₁, with controls.

FIG. 9 shows epitope specific lysis by CTL from HHD-A2 mice immunizedwith ED-B 29-38 peptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments of the invention disclosed herein provide compositions,methods of composition or vaccine design, and methods of treatmentrelated to the generation of a cellular immune response, preferably, a Tcell response and, more preferably, a CTL response, directed against theneovasculature of tumors. Such methods and compositions are particularlyuseful in the treatment and prevention of cancer. Other embodimentsrelate to composition evaluation models.

Compositions, Composition Design, and Treatment Using the Compositions

Embodiments of the invention relate to immunogenic compositions,including vaccines, for the generation of a cellular immune response,particularly a T cell response and specifically a CTL response, directedagainst tumor neovasculature (TuNV). “Tumor neovasculature” is broadlymeant to include any vasculature found in or around tumor masses,vasculature which supports or is necessary for tumor growth, and thelike. It should be noted, and one of skill in the art will appreciate,that although the discussion herein refers generally to the tumors andtumor neovasculature, the embodiments of the present invention also canbe applied to other conditions or disease states associated withinappropriate angiogenesis.

Until now the design of anti-tumor vaccines has concentrated on antigensexpressed by the malignant cells themselves. However, larger tumors arecomplex structures and not simply a homogeneous mass of cells. Allcells, particularly rapidly growing cells, need a supply of nutrients(oxygen, glucose, amino acids, etc.), as well as a means of removal ofmetabolic wastes, in order to remain metabolically active and intact.This is normally accomplished by the flow of blood and lymph through thevarious organs of the body. At a cellular level, the tissues of the bodyare permeated by a fine network of capillaries—tiny vessels throughwhich nutrients and waste products can be exchanged with the surroundingcells by diffusion. Diffusion is effective over relatively shortdistances. The capillary beds are so extensive that generally cells areat most located only a few cells away from a capillary. If a tumormerely grew by propagation of its malignant cells, soon those cells inthe interior of the mass would be unable to sustain themselves. In fact,the interiors of unvascularized tumors often contain necrotic tissue.Thus, in order to grow unchecked, tumors secrete factors that promotethe in-growth of new blood vessels, namely TuNV. Since the TuNVexpresses antigens differentiating it from other tissues, cancer can betreated with therapeutic compositions directed against the TuNV, insteadof directly targeting the cancerous cells themselves. Suitable TuNVantigens can include those that are expressed generally inneovasculature or preferentially by TuNV, for example.

In some embodiments of the invention the compositions can include, forexample, an epitopic peptide or peptides. Immune epitopes may beprovided embedded in epitope clusters and protein fragments.Housekeeping epitopes can be provided with the proper C-terminus. Inother embodiments of the invention the compositions can include nucleicacids capable of conferring expression of these epitopes on pAPC, forexample.

In preferred embodiments, the compositions can be administered directlyto the lymphatic system of a mammal being treated. This can be appliedto both polypeptide and nucleic acid based compositions. Administrationmethods of this type, and related technologies, are disclosed in U.S.patent application Ser. No. 09/380,534, filed on Sep. 1, 1999, and aContinuation-in-Part thereof, filed on Feb. 2, 2001; U.S. patentapplication Ser. No. 09/776,232, both entitled “A METHOD OF INDUCING ACTL RESPONSE,” which are incorporated by reference in their entirety.

In a preferred embodiment, destruction of the blood vessels in a tumorby action of a composition of the invention can eliminate all of thecells in a tumor. However, small tumors, including micrometastases, aretypically unvasculaturized. Additionally, unvascularized tumors thatinstead apparently rely on blood flow through channels penetrating thetumor mass have been reported (Maniotis, A. J., et al. Am. J. Pathol.155: 739-752, 1999). Thus in other embodiments, the compositions aregenerally effective as tumor control agents that may not eradicate allcancer cells. Accordingly, the invention provides tools for eliminatingtumors, controlling tumor growth, reducing tumor burden, improvingoverall clinical status, and the like. In some embodiments, it can bedesirable to combine these compositions with other treatments thattarget the cancerous cells directly. Additionally there is evidence thatthe vasculature in tumors can be mosaic in nature consisting of bothendothelial and cancer cells (Chang, Y. S., et al. Proc. Natl. Acad.Sci. USA 97:14608-14613, 2000). Thus, in some embodiments of theinvention a course of composition treatment can be followed byadministration of a bio- or chemotherapeutic agent. In a particularlypreferred embodiment, treatment can include administration of a TuAAdirected composition concurrent or subsequent to administration of theanti-TuNV composition.

As mentioned above, suitable TuNV antigens for the compositions caninclude those that are expressed generally in neovasculature orpreferentially by TuNV, for example. A variety of techniques fordiscovery of TuAA are known in the art. Examples of these techniquesinclude, without limitation, differential hybridization and subtractivehybridization, including use of microarrays; expression cloning; SAGE(serial analysis of gene expression); SEREX (serological identificationof antigens by recombinant expression cloning); in situ RT-PCT;immunohistochemistry (as was the case for PSMA); EST analysis; variouslyusing bulk, sectioned, and/or microdissected tissue; and the like.Utilization of these and other methods provides one of skill in the artthe techniques necessary to identify genes and gene products containedwithin a target cell that may be used as antigens of immunogeniccompositions. The techniques are applicable to TuAA discovery regardlessof whether the target cell is a cancer cell or an endothelial cell. Anyidentified antigen can be scrutinized for epitopes, which can be used inembodiments of the invention.

The endothelial cells making up the lining of the vasculature canexpress housekeeping proteasomes. Thus, compositions targetingendothelial cells can be comprised of peptides, or nucleic acidsconferring expression of the peptides, corresponding to the digestionproducts of the housekeeping proteasome (i.e. housekeeping epitopes).IFN-γ (gamma), secreted by activated cells of the immune system, caninduce expression of the immunoproteasome in the target cells.Generally, the immunoproteasome is constitutive in professional antigenpresenting cells (pAPC). Thus, it can be helpful to include immuneepitopes or epitope clusters in CTL-inducing compositions to ensure thatthere are CTL able to recognize the target cell regardless of the statethat the target cell is in. This can be particularly true withendothelial cells, which readily assume antigen presentation functions.These concepts are more fully explained in U.S. patent application Ser.No. 09/560,465, filed on Apr., 28, 2000; Ser. No. 10/005,905, filed onNov. 7, 2001; and a continuation thereof, U.S. application Ser. No.10/026,066, filed on Dec. 7, 2001, each of which is entitled “EPITOPESYNCHRONIZATION IN ANTIGEN PRESENTING CELLS,” and each of which ishereby incorporated by reference in its entirety.

As discussed above, the immunogenic compositions, including in preferredembodiments, vaccines, can include TuNV antigens and epitopes, forexample. The epitopes can include one or more housekeeping epitopesand/or one or more immune epitopes. Specific epitopes useful incompositions can be identified using the methods disclosed in U.S.patent application Ser. No. 09/561,074 entitled “METHOD OF EPITOPEDISCOVERY,” filed on Apr. 28, 2000. For example, peptide sequences thatare known or predicted to bind to some MHC restriction element can becompared to fragments produced by proteasomal digestion in order toidentify those that are co-C-terminal.

Examples of useful epitopes for the embodiments of the invention,including epitopes of ED-B and PSMA, are disclosed in a U.S. ProvisionalPatent Application No. 60/363,210, entitled “EPITOPE SEQUENCES,” filedon Mar. 7, 2002, and two U.S. Provisional Patent Applications, eachentitled “EPITOPE SEQUENCES;” Application No. 60/282,211, filed on Apr.6, 2001 and 60/337,017, filed on Nov. 7, 2001. Each of theseapplications is incorporated herein by reference in its entirety.

PSMA is one example of a TuAA that can be targeted in some embodiments.PSMA is expressed in the neovasculature of most tumor types, but not bythe vascular endothelium of normal tissues (Chang, S. M. et al., CancerRes. 59(13):3192-8,1999; Clin Cancer Res. 10:2674-81, 1999). PSMA is amembrane antigen, and as such, it may be possible to attackPSMA-expressing TuNV with monoclonal antibody (mAb). However, theeffectiveness of mAb in the treatment of cancer has proved to be moredifficult than initially anticipated. Moreover, as other antigens arediscovered to be associated with the TuNV, it is likely that many ofthem will prove not to be expressed at the vasculature surface, makingthem inaccessible to mAb attack.

T cells, particularly CTL, on the other hand, survey the expression ofinternal components of the cell through the process of majorhistocompatability complex (MHC)-restricted antigen presentation. Theparameters for determining the effectiveness of T cell-activatingvaccines and compositions against self-antigens are subtle. Some of thecritical features and parameters relating to appropriate epitopeselection are disclosed in U.S. patent application Ser. No. 09/560,465entitled “EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS,” filed onApr. 28, 2001; U.S. patent application Ser. No. 09/561,074 entitled“METHOD OF EPITOPE DISCOVERY,” filed on Apr. 28, 2001; and U.S. patentapplication Ser. No. 09/561,571 entitled “EPITOPE CLUSTERS,” filed onApr. 28, 2001. Features of DNA vaccine design promoting epitopesynchronization are disclosed in U.S. patent application Ser. No.09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OFTARGET-ASSOCIATED ANTIGENS,” filed on Apr. 28, 2001 and U.S. ProvisionalApplication No. 60/336,968 entitled “EXPRESSION VECTORS ENCODINGEPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN,”filed on Nov. 7, 2001. Particularly effective means of vaccine deliveryare described in U.S. patent application Ser. No. 09/380,534, filed onSep. 1, 1999, and a Continuation-in-Part thereof, U.S. patentapplication Ser. No. 09/776,232, filed on Feb. 2, 2001, both entitled “AMETHOD OF INDUCING A CTL RESPONSE.” Each of the above-mentionedreferences is incorporated herein by reference in its entirety.

Another example of a TuNV antigen that can be used in embodiments isfibronectin, preferably the ED-B domain. Fibronectin is subject todevelopmentally regulated alternative splicing, with the ED-B domainbeing encoded by a single exon that is used primarily in oncofetaltissues. Matsuura, H. and S. Hakomori Proc. Natl. Acad. Sci. USA82:6517-6521, 1985; Carnemolla, B. et al. J. Cell Biol. 108:1139-1148,1989; Loridon-Rosa, B. et al. Cancer Res. 50:1608-1612, 1990; Nicolo, G.et al. Cell Differ. Dev. 32:401-408, 1990; Borsi, L. et al. Exp. CellRes. 199:98-105, 1992; Oyama, F. et al. Cancer Res. 53:2005-2011, 1993;Mandel, U. et al. APMIS 102:695-702, 1994; Famoud, M. R. et al. Int. J.Cancer 61:27-34, 1995; Pujuguet, P. et al. Am. J. Pathol. 148:579-592,1996; Gabler, U. et al. Heart 75:358-362, 1996; Chevalier, X. Br. J.Rheumatol. 35:407-415, 1996; Midulla, M. Cancer Res. 60:164-169, 2000.

The ED-B domain is also expressed in fibronectin of the neovasculatureKaczmarek, J. et al. Int. J. Cancer 59:11-16, 1994; Castellani, P. etal. Int. J. Cancer 59:612-618, 1994; Neri, D. et al. Nat. Biotech.15:1271-1275, 1997; Karelina, T. V. and A. Z. Eisen Cancer Detect. Prev.22:438-444, 1998; Tarli, L. et al. Blood 94:192-198, 1999; Castellani,P. et al. Acta Neurochir. (Wien) 142:277-282, 2000. As an oncofetaldomain, the ED-B domain is commonly found in the fibronectin expressedby neoplastic cells, in addition to being expressed by the TuNV.Therefore, CTL-inducing compositions targeting the ED-B domain canexhibit two mechanisms of action: direct lysis of tumor cells, anddisruption of tumor blood supply through destruction of the TuNV.

It should be noted that expression of the fibronectin ED-B domain hasbeen reported in both tumor-associated and normal neovasculature(Castellani, P. et al. Int. J. Cancer 59:612-618, 1994). Thus,compositions based on it, or similarly expressed antigens, can beeffective against other conditions associated with inappropriateangiogenesis. Further, as CTL activity can decay rapidly afterwithdrawal of the composition, interference with normal angiogenesis canbe minimal.

Other targets for the immunogenic compositions include growth factorreceptors, including those associated with vascular cells. One suchexample is the vascular endothelial growth factor receptor 2 (VEGFR2).U.S. Pat. No. 6,342,221 includes discussion of VEGF and VEGFR2, and ishereby incorporated by reference in its entirety. One of skill in theart will appreciate that any other antigen or protein associated withvascular cells can be a target for the immunogenic compositions,including those that are presently known and those yet to be identified.

Animal Models, Methods of Making the Models, and Composition Evaluation

Compositions designed based upon the preceding considerations areeffective against the various targets. However, additional evaluationcan be easily performed at any time, but preferably in a pre-clinicalsetting. For example, such evaluation can be used in order to furtheraid in composition design. Other embodiments of the invention relate tomethods of evaluating the immunogenic compositions. The compositions ofthe present invention can be easily evaluated by one of skill in the artusing animal models for composition evaluation. For example, followingthe routine procedures below, one of skill in the art can evaluate TuNVcompositions quickly and efficiently. Thus, using the models or guidancedescribed herein, one of skill in the art can evaluate any TuNVcomposition for any TuNV antigen with little or no experimentation.Further embodiments relate to methods of making the animal researchmodels. Other embodiments relate to the research model animals. Theseembodiments are set forth more fully below.

Xenotransplanted Human Vasculature-Based Model

Some embodiments relate to a model system for studying the mechanisms ofhuman microvessel formation. For example, in some embodiments, the modelsystem can be used for preclinical evaluation of compositions. The modelinvolves the subcutaneous implantation of telomerase-transformed humandermal microvascular endothelial cells (HDMEC) mixed with MATRIGEL(Becton Dickinson) into SCID mice. Subcutaneous implantation oftelomerase-transformed HCMEC is described in Yang, J. et al. NatureBiotech 19:219-224, 2001, which is hereby incorporated by reference inits entirety. T cells activated by the compositions of this inventioncan be adoptively transferred, for example, into such implanted mice,and the ability of the T cells to destroy, or prevent the formation of,such human microvessels can be assessed. In other embodiments, the mousecan be directly vaccinated and evaluated. Also, in still furtherembodiments, the model system can be further adapted for testingcompositions effective in non-human species by substituting DMEC fromother species and species-matched telomerase, and by using analogousreagents to those described below for the human system.

The MHC-restriction elements presenting the epitopes of the compositionbeing tested, preferably, are shared by the HDMEC line implanted intothe mice. The T cells can be derived from in vitro immunization of humanT cells, or by immunization of HLA-transgenic mice (procedures for whichare well known in the art and examples of which are provided in theabove incorporated patent applications). Use of T cells generated inHLA-transgeneic mice allows matching of genetic backgrounds between theadoptively transferred T cells and the host, thereby reducing thepossibility of allogeneic or xenogeneic reactions that might complicateinterpretation of the results. However, depending on the mouse strainsavailable, this might require cross-breeding to get the HLA-transgeneand SCID phenotype on the same genetic background. In the alternative,the donor T cells (human or murine) can be subjected to one or morerounds of in vitro stimulation to enrich for the desired population orestablish a clone, and thereby similarly avoid undesired reactivities.

Techniques for in vitro immunization are know in the art, for example,Stauss et al., Proc. Natl. Acad. Sci. USA 89:7871-7875, 1992; Salgalleret al. Cancer Res. 55:4972-4979, 1995; Tsai et al., J. Immunol.158:1796-1802, 1997; and Chung et al., J Immunother. 22:279-287, 1999.Once generated, whether in vivo or in vitro, sufficient numbers of suchT cells can be obtained by expansion in vitro through stimulation withthe compositions of this invention and/or cytokines (see for exampleKurokawa, T. et al., Int. J. Cancer 91:749-746, 2001) or other mitogens.These T cells can constitute a clone or a polyclonal populationrecognizing one or more epitopes. In preferred embodiments, on the orderof 10⁵ to 10⁸ cells are transferred for adoptive transfer experiments inmice. (See for example Drobyski, W. R. et al. Blood 97:2506-2513, 2001;Seeley B. M. et al. Otolaryngol. Head Neck Surg. 124:436-441, 2001;Kanwar, J. R. et al. Cancer Res. 61:1948-1956, 2001). Clones andotherwise more enriched populations generally require the transfer offewer cells.

Transfer of the T cells can take place shortly before, concurrent with,or subsequent to implantation or establishment of the HDMEC. Parametersthat can be assessed to evaluate effectiveness of the compositionsinclude vessel formation, changes in vessel density, and ability tocarry mouse blood (as described in Yang et al.), and the like.Assessment can be carried out as early as one week, and at least as longas 6 weeks, after implantation of telomerase-transformed HDMEC,preferably after 2 weeks; and from a day to more than 6 weeks after Tcell transfer, preferably after 1 to 3 weeks. Generally, assessment caninclude comparison of mice receiving T cells reactive with the targetantigen with mice receiving naïve (including sham-immunized), orirrelevant epitope-reactive T cells.

Relevant antigens can be expressed generally in neovasculature orpreferentially by TuNV. Expression can be confirmed by a variety oftechniques known in the art, including immunohistochemistry and RT-PCR.For example, tumor cells can be implanted along with the HDMEC. This canresult in inducing expression of antigens preferentially expressed byTuNV. In one example, this can be accomplished by implanting a block oftumor tissue adjacent to the HDMEC-containing MATRIGEL implant,injecting tumor cells at the site of the implant, implanting tumorcell-containing MATRIGEL adjacent to the HDMEC-containing MATRIGELimplant, incorporating both tumor cells and HDMEC into the same MATRIGELimplant or by any other suitable method. As discussed above, in someembodiments, tumor cells can be implanted along with vascular cells. Theanimals so made, can be used as research models. Additional variationswill be apparent to one of skill in the art.

HLA-transgenic Animal Model

For antigens that are conserved, in sequence and/or expression profile,between human and the model species, HLA-transgenic strains allowanother approach, namely vaccination of the model animal to combat asyngeneic tumor. The ED-B domain of fibronectin provides such anopportunity, as it is a marker of angiogenesis and has identical aminoacid sequence in both humans and mice (Nilsson, F. et al. Cancer Res.61:711-716, 2001). Moreover, spontaneous tumor tissue from a strain ofHLA-A2 transgenic mice has been isolated and propagated. Epitopediscovery and selection, and composition design and delivery for CTLinducing compositions are discussed above.

The tumor cell line, M1, is derived from a spontaneous salivaryglandular cystadenocarcinoma. The M1 tumor cell line and methods ofusing the same is disclosed in U.S. Provisional Application No.60/363,131, Mar. 7, 2002, entitled “AN HLA-TRANSGENIC MURINE TUMOR CELLLINE,” which is hereby expressly incorporated by reference in itsentirety. The tumor cell line, can arise in individuals of the HHD-A2transgenic mouse strain of S. Pascolo et al. (J. Exp. Med.185:2043-2051, 1997). These mice express a single monochain class Imolecule comprising human β (beta)₂-microglobulin, and α1 (alpha-1), andα2 (alpha-2) domains of HLA-A2.1 with the balance of the moleculederived from the murine class I molecule H2 D^(b). Blocks of tumor canbe transplanted into new individuals where the tumor will re-grow,commonly within 1-3 weeks, with 3 mm blocks growing to 3 cm.Alternatively, tumor tissue can be disaggregated and the tumor cellsgrown in vitro. Upon harvest, the tumor cells can be injectedsubcutaneously into the neck or abdomen (2.5×10⁶ cells for 1-3successive days), to result in a visible tumor in approximately 5-12weeks for early passage cells. After the cells have become betteradapted to growth in vitro, single injections of 1×10⁶ to 1×10⁷ cellslead to visible tumor in ten days. Generally, the initial tumorconsistently occurs in the vicinity of the salivary glands, butsecondary tumors can also occur in a variety of locations, includingkidney, lung, liver, and abdominal muscle.

To evaluate the efficacy of a composition, it can be administeredbefore, concurrent with, or subsequent to establishment of the tumor,depending on the expected mechanism of the composition. For therapeuticcompositions intended to be used with some sort of debulking technique(e.g. surgery), concurrent administration can be appropriate. The betterestablished the tumor is before treatment begins, the more stringent thetest.

Both animal evaluation models have been described for the testing ofhuman compositions. However, application to veterinary compositions isanalogous, requiring only the substitution of species-matchedendothelial cells, MHC, TuAA, etc.

All patents, patent applications, and publications referred to hereinare hereby incorporated by reference in their entirety.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLES Example 1

A preclinical study was carried out using the already identifiedantigens PSMA and ED-B disclosed herein. The results of the studyrevealed excellent candidate epitopes. See table 9 below.

Example 1.1

Cluster Analysis (PSMA₁₆₃₋₁₉₂)

A peptide, AFSPQGMPEGDLVYVNYARTEDFFKLERDM, PSMA₁₆₃-192, (SEQ ID NO. 3),containing an A1 epitope cluster from prostate specific membraneantigen, PSMA₁₆₈₋₁₉₀ (SEQ ID NO. 4) was synthesized using standardsolid-phase F-moc chemistry on a 433A ABI Peptide synthesizer. Afterside chain deprotection and cleavage from the resin, peptide firstdissolved in formic acid and then diluted into 30% Acetic acid, was runon a reverse-phase preparative HPLC C4 column at following conditions:linear AB gradient (5% B/min) at a flow rate of 4 ml/min, where eluent Ais 0.1% aqueous TFA and eluent B is 0.1% TFA in acetonitrile. A fractionat time 16.642 min containing the expected peptide, as judged by massspectrometry, was pooled and lyophilized. The peptide was then subjectedto proteasome digestion and mass spectrum analysis essentially asdescribed above. Prominent peaks from the mass spectra are summarized inTable 1. TABLE 1 PSMA₁₆₃₋₁₉₂ Mass Peak Identification. CALCU- SEQ LATEDID MASS NO. PEPTIDE SEQUENCE (MH⁺) 110 163-177 AFSPQGMPEGDLVYV 1610.0111 178-189                NYARTEDFFKLE 1533.68 112 170-189       PEGDLVYVNYARTEDFFKLE 2406.66 113 178-191               NYARTEDFFKLERD 1804.95 114 170-191       PEGDLVYVNYARTEDFFKLERD 2677.93 115 178-192               NYARTEDFFKLERDM 1936.17 116 163-176 AFSPQGMPEGDLVY1511.70 117 177-192               VNYARTEDFFKLERDM 2035.30 118 163-179AFSPQGMPEGDLVYVNY 1888.12 119 180-192                  ARTEDFFKLERDM1658.89 120 163-183 AFSPQGMPEGDLVYVNYARTE 2345.61 121 184-192                     DFFKLERDM 1201.40 122 176-192             YVNYARTEDFFKLERDM 2198.48 123 167-185    QGMPEGDLVYVNYARTEDF 2205.41 124 178-186                NYARTEDFF1163.22Boldface sequences correspond to peptides predicted to bind to MHC, seeTable 2.N-Terminal Pool Sequence Analysis

One aliquot at one hour of the proteasomal digestion was subjected toN-nal terminal amino acid sequence analysis by an ABI 473A ProteinSequencer (Applied Biosystems, Foster City, Calif.). Determination ofthe sites and efficiencies of cleavage was based on consideration of thesequence cycle, the repetitive yield of the protein sequencer, and therelative yields of amino acids unique in the analyzed sequence. That isif the unique (in the analyzed sequence) residue X appears only in thenth cycle a cleavage site exists n-1 residues before it in theN-terminal direction. In addition to helping resolve any ambiguity inthe assignment of mass to sequences, these data also provide a morereliable indication of the relative yield of the various fragments thandoes mass spectrometry.

For PSMA₁₆₃₋₁₉₂ (SEQ ID NO. 3) this pool sequencing supports a singlemajor cleavage site after V₁₇₇ and several minor cleavage sites,particularly one after Y₁₇₉. Reviewing the results presented in FIGS.1A-C reveals the following:

-   -   S at the 3^(rd) cycle indicating presence of the N-terminus of        the substrate.    -   Q at the 5^(th) cycle indicating presence of the N-terminus of        the substrate.    -   N at the 1^(st) cycle indicating cleavage after V₁₇₇.    -   N at the 3^(rd) cycle indicating cleavage after V₁₇₅. Note the        fragment 176-192 in Table 1.    -   T at the 5^(th) cycle indicating cleavage after V₁₇₇.    -   T at the 1^(st)-3^(rd) cycles, indicating increasingly common        cleavages after R₁₈₁, A₁₈₀ and Y₁₇₉. Only the last of these        correspond to peaks detected by mass spectrometry; 163-179 and        180-192, see Table 1. The absence of the others can indicate        that they are on fragments smaller than were examined in the        mass spectrum.    -   K at the 4^(th), 8^(th), and 10^(th) cycles indicating cleavages        after E₁₈₃, Y₁₇₉, and V₁₇₇, respectively, all of which        correspond to fragments observed by mass spectroscopy. See Table        1.    -   A at the 1^(st) and 3^(rd) cycles indicating presence of the        N-terminus of the substrate and cleavage after V₁₇₇,        respectively.    -   P at the 4^(th) and 8^(th) cycles indicating presence of the        N-terminus of the substrate.    -   G at the 6^(th) and 10^(th) cycles indicating presence of the        N-terminus of the substrate.    -   M at the 7^(th) cycle indicating presence of the N-terminus of        the substrate and/or cleavage after F₁₈₅.    -   M at the 15^(th) cycle indicating cleavage after V₁₇₇.    -   The 1^(st) cycle can indicate cleavage after D₁₉₁, see Table 1.    -   R at the 4^(th) and 13^(th) cycle indicating cleavage after        V₁₇₇.    -   R at the 2^(nd) and 11^(th) cycle indicating cleavage after        Y₁₇₉.    -   V at the 2^(nd), 6^(th), and 13^(th) cycle indicating cleavage        after V₁₇₅, M₁₆₉ and presence of the N-terminus of the        substrate, respectively. Note fragments beginning at 176 and 170        in Table 1.    -   Y at the 1^(st), 2^(nd), and 14^(th) cycles indicating cleavage        after V₁₇₅, V₁₇₇, and presence of the N-terminus of the        substrate, respectively.    -   L at the 11^(th) and 12^(th) cycles indicating cleavage after        V₁₇₇, and presence of the N-terminus of the substrate,        respectively, is the interpretation most consistent with the        other data. Comparing to the mass spectrometry results we see        that    -   L at the 2^(nd), 5^(th), and 9^(th) cycles is consistent with        cleavage after F₁₈₆, E₁₈₃ or M₁₆₉, and Y₁₇₉, respectively. See        Table 1.        Epitope Identification

Fragments co-C-terminal with 8-10 amino acid long sequences predicted tobind HLA by the SYFPEITHI or NIH algorithms were chosen for furtheranalysis. The digestion and prediction steps of the procedure can beusefully practiced in any order. Although the substrate peptide used inproteasomal digest described here was specifically designed to include apredicted HLA-A1 binding sequence, the actual products of digestion canbe checked after the fact for actual or predicted binding to other MHCmolecules. Selected results are shown in Table 2. TABLE 2 Predicted HLAbinding by proteasomally generated fragments I. SEQ ID NO II. PEPTIDEHLA SYFPEITHI NIH  5 & (6) (G)MPEGDLVYV A*0201 17(27) (2605) B*0702 20<5 B*5101 22 314  7 & (8) (Q)GMPEGDLVY A1 24(26) <5 A3 16(18) 36 B*270517 25  9 MPEGDLVY B*5101 15 NP† 10 & (11) (P)EGDLVYVNY A1 27(15) 12 A2623(17) NP 12 LVYVNYARTE A3 21 <5 13 & (14) (Y)VNYARTEDF A26 (20) NP B*0815 <5 B*2705 12 50 15 NYARTEDFF A24 NP† 100 Cw*0401 NP 120 16 YARTEDFFB*08 16 <5 17 RTEDFFKLE A1 21 <5 A26 15 NP†No predictionHLA-A*0201 Binding Assay:

Binding of the candidate epitope PSMA₁₆₈₋₁₇₇, GMPEGDLVYV, (SEQ ID NO. 6)to HLA-A2.1 was assayed using a modification of the method of Stauss etal., (Proc Natl Acad Sci USA 89(17):7871-5 (1992)). Specifically, T2cells, which express empty or unstable MHC molecules on their surface,were washed twice with Iscove's modified Dulbecco's medium (IMDM) andcultured overnight in serum-free AIM-V medium (Life Technologies, Inc.,Rockville, Md.) supplemented with human β2-microglobulin at 3 μg/ml(Sigma, St. Louis, Mo.) and added peptide, at 800, 400, 200, 100, 50,25, 12.5, and 6.25 μg/ml.in a 96-well flat-bottom plate at 3×10⁵cells/200 μl/well. Peptide was mixed with the cells by repipeting beforedistributing to the plate (alternatively peptide can be added toindividual wells), and the plate was rocked gently for 2 minutes.Incubation was in a 5% CO₂ incubator at 37° C. The next day the unboundpeptide was removed by washing twice with serum free RPMI medium and asaturating amount of anti-class I HLA monoclonal antibody, fluoresceinisothiocyanate (FITC)-conjugated anti-HLA A2, A28 (One Lambda, CanogaPark, Calif.) was added. After incubation for 30 minutes at 4° C., cellswere washed 3 times with PBS supplemented with 0.5% BSA, 0.05% (w/v)sodium azide, pH 7.4-7.6 (staining buffer). (Alternatively W6/32 (Sigma)can be used as the anti-class I HLA monoclonal antibody the cells washedwith staining buffer and then incubated with fluorescein isothiocyanate(FITC)-conjugated goat F(ab′) antimouse-IgG (Sigma) for 30 min at 4° C.and washed 3 times as before.) The cells were resuspended in 0.5 mlstaining buffer. The analysis of surface HLA-A2.1 molecules stabilizedby peptide binding was performed by flow cytometry using a FACScan(Becton Dickinson, San Jose, Calif.). If flow cytometry is not to beperformed immediately the cells can be fixed by adding a quarter volumeof 2% paraformaldehyde and storing in the dark at 4° C.

As seen in FIG. 2, this epitope exhibits significant binding at evenlower concentrations than the positive control peptides. The Melan-Apeptide used as a control in this assay (and throughout thisdisclosure), ELAGIGILTV (SEQ ID NO: 106), is actually a variant of thenatural sequence (EAAGIGILTV; SEQ ID NO: 107)) and exhibits a highaffinity in this assay. The known A2.1 binder FLPSDYFPSV (HBV₁₈₋₂₇; SEQID NO: 107) was also used as a positive control. An HLA-B44 bindingpeptide, AEMGKYSFY (SEQ ID NO: 109), was used as a negative control. Thefluorescence obtained from the negative control was similar to thesignal obtained when no peptide was used in the assay. Positive andnegative control peptides were chosen from Table 18.3.1 in CurrentProtocols in Immunology p. 18.3.2, John Wiley and Sons, New York, 1998.

Example 1.2

Cluster Analysis (PSMA₂₈₁₋₃₁₀).

Another peptide, RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG, PSMA₂₈₁₋₃₁₀, (SEQ IDNO. 18), containing an A1 epitope cluster from prostate specificmembrane antigen, PSMA₂₈₃₋₃₀₇ (SEQ ID NO. 19), was synthesized usingstandard solid-phase F-moc chemistry on a 433A ABI Peptide synthesizer.After side chain deprotection and cleavage from the resin, peptide inddH2O was run on a reverse-phase preparative HPLC C18 column atfollowing conditions: linear AB gradient (5% B/min) at a flow rate of 4ml/min, where eluent A is 0.1% aqueous TFA and eluent B is 0.1% TFA inacetonitrile. A fraction at time 17.061 min containing the expectedpeptide as judged by mass spectrometry, was pooled and lyophilized. Thepeptide was then subjected to proteasome digestion and mass spectrumanalysis essentially as described above. Prominent peaks from the massspectra are summarized in Table 3. TABLE 3 PSMA₂₈₁₋₃₁₀ Mass PeakIdentification. CALCU- SEQ LATED ID MASS NO. PEPTIDE SEQUENCE (MH⁺) 125281-297 RGIAEAVGLPSIPVHPI* 1727.07 126 286-297      AVGLPSIPVHPI**1200.46 127 287-297       VGLPSIPVHPI 1129.38 128 288-297       GLPSIPVHPI^(†) 1030.25 129 298-310                GYYDAQKLLEKMG≠1516.5 130 298-305                  GYYDAQKL§ 958.05 131 281-305RGIAEAVGLPSIPVHPIGYYDAQKL 2666.12 132 281-307RGIAEAVGLPSIPVHPIGYYDAQKLLE 2908.39 133 286-307     AVGLPSIPVHPIGYYDAQKLLE¶ 2381.78 134 287-307      VGLPSIPVHPIGYYDAQKLLE 2310.70 135 288-307       GLPSIPVHPIGYYDAQKLLE# 2211.57 136 281-299 RGIAEAVGLPSIPVHPIGY1947 137 286-299      AVGLPSIPVHPIGY 1420.69 138 287-299      VGLPSIPVHPIGY 1349.61 139 288-299        GLPSIPVHPIGY 1250.48 140287-310       VGLPSIPVHPIGYYDAQKLLEKMG 2627.14 141 288-310       GLPSIPVHPIGYYDAQKLLEKMG 2528.01Boldface sequences correspond to peptides predicted to bind to MHC, seeTable 4.*By mass alone this peak could also have been 296-310 or 288-303.**By mass alone this peak could also have been 298-307. Combination ofHPLC and mass spectrometry show that at some later time points this peakis a mixture of both species.^(†)By mass alone this peak could also have been 289-298.≠By mass alone this peak could also have been 281-295 or 294-306.§By mass alone this peak could also have been 297-303.¶By mass alone this peak could also have been 285-306.#By mass alone this peak could also have been 288-303.None of these alternate assignments are supported N-terminal poolsequence analysis.N-Terminal Pool Sequence Analysis

One aliquot at one hour of the proteasomal digestion (see Example 3 part3 above) was subjected to N-terminal amino acid sequence analysis by anABI 473A Protein Sequencer (Applied Biosystems, Foster City, Calif.).Determination of the sites and efficiencies of cleavage was based onconsideration of the sequence cycle, the repetitive yield of the proteinsequencer, and the relative yields of amino acids unique in the analyzedsequence. That is if the unique (in the analyzed sequence) residue Xappears only in the nth cycle a cleavage site exists n-1 residues beforeit in the N-terminal direction. In addition to helping resolve anyambiguity in the assignment of mass to sequences, these data alsoprovide a more reliable indication of the relative yield of the variousfragments than does mass spectrometry.

For PSMA₂₈₁₋₃₁₀ (SEQ ID NO. 18) this pool sequencing supports two majorcleavage sites after V₂₈₇ and 1297 among other minor cleavage sites.Reviewing the results presented in FIG. 3 reveals the following:

-   -   S at the 4^(th) and 11^(th) cycles indicating cleavage after        V₂₈₇ and presence of the N-terminus of the substrate,        respectively.    -   H at the 8^(th) cycle indicating cleavage after V₂₈₇. The lack        of decay in peak height at positions 9 and 10 versus the drop in        height present going from 10 to 11 can suggest cleavage after        A₂₈₆ and E₂₈₅ as well, rather than the peaks representing        latency in the sequencing reaction.    -   D at the 2^(nd), 4^(th), and 7^(th) cycles indicating cleavages        after Y₂₉₉, 1297, and V₂₉₄, respectively. This last cleavage is        not observed in any of the fragments in Table 4 or in the        alternate assignments in the notes below.    -   Q at the 6^(th) cycle indicating cleavage after 1297.    -   M at the 10^(th) and 12^(th) cycle indicating cleavages after        Y₂₉₉ and I₂₉₇, respectively.        Epitope Identification

Fragments co-C-terminal with 8-10 amino acid long sequences predicted tobind HLA by the SYFPEITHI or NIH algorithms were chosen for furtherstudy. The digestion and prediction steps of the procedure can beusefully practiced in any order. Although the substrate peptide used inproteasomal digest described here was specifically designed to include apredicted HLA-A1 binding sequence, the actual products of digestion canbe checked after the fact for actual or predicted binding to other MHCmolecules. Selected results are shown in Table 4. TABLE 4 Predicted HLAbinding by proteasomally generated fragments: PSMA₂₈₁₋₃₁₀ III. SEQ IDNO. IV. PEPTIDE HLA SYFPEITHI NIH 20 & (21) (G)LPSIPVHPI A*0201 16(24)(24) B*0702/B7 23 12 B*5101 24 572 Cw*0401 NP† 20 22 & (23) (P)IGYYDAQKLA*0201 (16) <5 A26 (20) NP B*2705 16 25 B*2709 15 NP B*5101 21 57Cw*0301 NP 24 24 & (25) (P)SIPVHPIGY A1 21(27) <5 A26 22 NP A3 16 <5 26IPVHPIGY B*5101 16 NP 27 YYDAQKLLE A1 22 <5†No predictionAs seen in Table 4, N-terminal addition of authentic sequence toepitopes can often generate still useful, even better epitopes, for thesame or different MHC restriction elements. Note for example the pairingof (G)LPSIPVHPI with HLA-A*0201, where the 10-mer can be used as avaccine useful with several MHC types by relying on N-terminal trimmingto create the epitopes for HLA-B7, -B*5101, and Cw*0401.HLA-A*0201 Binding Assay:

HLA-A*0201 binding studies were preformed with PSMA₂₈₈₋₂₉₇, GLPSIPVHPI,(SEQ ID NO. 21) essentially as described in Example 1.1 above. As seenin FIG. 2, this epitope exhibits significant binding at even lowerconcentrations than the positive control peptides.

Example 1.3

Cluster Analysis (PSMA₄₅₄₋₄₈₁).

Another peptide, SSIEGNYTLRVDCTPLMYSLVHLTKEL, PSMA₄₅₄₋₄₈₁, (SEQ ID NO.28) containing an epitope cluster from prostate specific membraneantigen, was synthesized by MPS (purity >95%) and subjected toproteasome digestion and mass spectrum analysis as described above.Prominent peaks from the mass spectra are summarized in Table 5. TABLE 5PSMA₄₅₄₋₄₈₁ Mass Peak Identification. SEQ ID MS PEAK CALCULATED NO.(measured) PEPTIDE SEQUENCE MASS (MH⁺) 142 1238.5 454-464 SSIEGNYTLRV1239.78 143 1768.38 ± 0.60 454-469 SSIEGNYTLRVDCTPL 1768.99 144 1899.8454-470 SSIEGNYTLRVDCTPLM 1900.19 145 1097.63 ± 0.91 463-471         RVDCTPLMY 1098.32 146 2062.87 ± 0.68 454-471*SSIEGNYTLRVDCTPLMY 2063.36 147 1153 472-481** SLVHNLTKEL 1154.36 1481449.93 ± 1.79 470-481 MYSLVHNLTKEL 1448.73Boldface sequence correspond to peptides predicted to bind to MHC, seeTable 6.*On the basis of mass alone this peak could equally well be assigned tothe peptide 455-472 however proteasomal removal of just the N-terminalamino acid is considered unlikely. If the issue were important it couldbe resolved by N-terminal sequencing.**On the basis of mass this fragment might also represent 455-464.Epitope Identification

Fragments co-C-terminal with 8-10 amino acid long sequences predicted tobind HLA by the SYFPEITHI or NIH algorithms were chosen for furtherstudy. The digestion and prediction steps of the procedure can beusefully practiced in any order. Although the substrate peptide used inproteasomal digest described here was specifically designed to includepredicted HLA-A2.1 binding sequences, the actual products of digestioncan be checked after the fact for actual or predicted binding to otherMHC molecules. Selected results are shown in Table 6. TABLE 6 PredictedHLA binding by proteasomally generated fragments V. SEQ ID NO VI.PEPTIDE HLA SYFPEITHI NIH 29 & (30) (S)IEGNYTLRV A1 (19) <5 A*020116(22) <5 31 EGNYTLRV B*5101 15 NP† 32 & (33) (Y)TLRVDCTPL A*0201 20(18)(5) A26 16(18) NP B7 14 40 B8 23 <5 B*2705 12 30 Cw*0301 NP (30) 34LRVDCTPLM B*2705 20 600 B*2709 20 NP 35 & (36) (L)RVDCTPLMY A1 32(22)125(13.5) A3 25 <5 A26 22 NP B*2702 NP (200) B*2705 13(NP) (1000)†No prediction

As seen in Table 6, N-terminal addition of authentic sequence toepitopes can often generate still useful, even better epitopes, for thesame or different MHC restriction elements. Note for example the pairingof (L)RVDCTPLMY (SEQ ID NOS 35 and (36)) with HLA-B*2702/5, where the10-mer has substantial predicted halftimes of dissociation and theco-C-terminal 9-mer does not. Also note the case of SIEGNYTLRV (SEQ IDNO 30) a predicted HLA-A*0201 epitope which can be used as a vaccineuseful with HLA-B*5101 by relying on N-terminal trimming to create theepitope.

HLA-A*0201 Binding Assay

HLA-A*0201 binding studies were preformed, essentially as described inExample 1.1 above, with PSMA₄₆₀₋₄₆₉, YTLRVDCTPL, (SEQ ID NO.33). As seenin FIG. 4, this epitope was found to bind HLA-A2.1 to a similar extentas the known A2.1 binder FLPSDYFPSV (HBV₁₈₋₂₇; SEQ ID NO: 108) used as apositive control. Additionally, PSMA₄₆₁₋₄₆₉, (SEQ ID NO. 32) bindsnearly as well.

ELISPOT analysis: PSMA₄₆₃₋₄₇₁ (SEQ ID NO. 35)

The wells of a nitrocellulose-backed microtiter plate were coated withcapture antibody by incubating overnight at 4° C. using 50 μl/well of 4μg/ml murine anti-human □-IFN monoclonal antibody in coating buffer (35mM sodium bicarbonate, 15 mM sodium carbonate, pH 9.5). Unbound antibodywas removed by washing 4 times 5 min. with PBS. Unbound sites on themembrane then were blocked by adding 200 μl/well of RPMI medium with 10%serum and incubating 1 hr. at room temperature. Antigen stimulated CD8⁺T cells, in 1:3 serial dilutions, were seeded into the wells of themicrotiter plate using 100 μl/well, starting at 2×10⁵ cells/well. (Priorantigen stimulation was essentially as described in Scheibenbogen, C. etal. Int. J. Cancer 71:932-936, 1997; which is incorporated herein byreference in its entirety.) PSMA₄₆₂₋₄₇₁ (SEQ ID NO. 36) was added to afinal concentration of 10 μg/ml and IL-2 to 100 U/ml and the cellscultured at 37° C. in a 5% CO₂, water-saturated atmosphere for 40 hrs.Following this incubation the plates were washed with 6 times 200μl/well of PBS containing 0.05% Tween-20 (PBS-Tween). Detectionantibody, 50 μl/well of 2 g/ml biotinylated murine anti-human □-IFNmonoclonal antibody in PBS+10% fetal calf serum, was added and the plateincubated at room temperature for 2 hrs. Unbound detection antibody wasremoved by washing with 4 times 200 μl of PBS-Tween. 100 μl ofavidin-conjugated horseradish peroxidase (Pharmingen, San Diego, Calif.)was added to each well and incubated at room temperature for 1 hr.Unbound enzyme was removed by washing with 6 times 200 μl of PBS-Tween.Substrate was prepared by dissolving a 20 mg tablet of 3-amino9-ethylcoarbasole in 2.5 ml of N,N-dimethylformamide and adding thatsolution to 47.5 ml of 0.05 M phosphate-citrate buffer (pH 5.0). 25 μlof 30% H₂O₂ was added to the substrate solution immediately beforedistributing substrate at 100 μl/well and incubating the plate at roomtemperature. After color development (generally 15-30 min.), thereaction was stopped by washing the plate with water. The plate was airdried and the spots counted using a stereomicroscope.

FIG. 5 shows the detection of PSMA₄₆₃₋₄₇₁ (SEQ ID NO. 35)-reactiveHLA-A1⁺ CD8⁺ T cells previously generated in cultures of HLA-A1⁺ CD8⁺ Tcells with autologous dendritic cells plus the peptide. No reactivity isdetected from cultures without peptide (data not shown). In this case itcan be seen that the peptide reactive T cells are present in the cultureat a frequency between 1 in 2.2×10⁴ and 1 in 6.7×10⁴. That this is trulyan HLA-A1-restricted response is demonstrated by the ability ofanti-HLA-A1 monoclonal antibody to block □-IFN production; see FIG. 6.

Example 1.4

Cluster Analysis (PSMA₆₅₃₋₆₈₇).

Another peptide, FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY PSMA₆₅₃₋₆₈₇, (SEQID NO. 37) containing an A2 epitope cluster from prostate specificmembrane antigen, PSMA₆₆₀₋₆₈₁ (SEQ ID NO. 38), was synthesized by MPS(purity>95%) and subjected to proteasome digestion and mass spectrumanalysis as described above. Prominent peaks from the mass spectra aresummarized in Table 7. TABLE 7 PSMA₆₅₃₋₆₈₇ Mass Peak Identification. SEQID MS PEAK CALCULATED NO. (measured) PEPTIDE SEQUENCE MASS (MH⁺) 149  906.17 ± 0.65   681-687** LPDRPFY 908.05 150 1287.73 ± 0.76  677-687** DPLGLPDRPFY 1290.47 151  1400.3 ± 1.79 676-687  IDPLGLPDRPFY 1403.63 152  1548.0 ± 1.37 675-687 FIDPLGLPDRPFY 1550.80153  1619.5 ± 1.51   674-687** AFIDPLGLPDRPFY 1621.88 154 1775.48 ± 1.32 673-687* RAFIDPLGLPDRPFY 1778.07 155  2440.2 ± 1.3 653-672FDKSNPIVLRMMNDQLMFLE 2442.932313.82 156 1904.63 ± 1.56  672-687*ERAFIDPLGLPDRPFY 1907.19 157  2310.6 ± 2.5 653-671 FDKSNPIVLRMMNQLMFL2313.82 158  2017.4 ± 1.94 671-687 LERAFIDPLGLPDRPFY 2020.35 159 2197.43± 1.78 653-670 FDKSNPIVLRMMNDQLMF 2200.66Boldface sequence correspond to peptides predicted to bind to MHC, seeTable 7.*On the basis of mass alone this peak could equally well be assigned toa peptide beginning at 654, however proteasomal removal of just theN-terminal amino acid is considered unlikely. If the issue wereimportant it could be resolved by N-terminal sequencing.**On the basis of mass alone these peaks could have been assigned tointernal fragments, but given the overall pattern of digestion it wasconsidered unlikely.Epitope Identification

Fragments co-C-terminal with 8-10 amino acid long sequences predicted tobind HLA by the SYFPEITHI or NIH algorithms were chosen for furtherstudy. The digestion and prediction steps of the procedure can beusefully practiced in any order. Although the substrate peptide used inproteasomal digest described here was specifically designed to includepredicted HLA-A2.1 binding sequences, the actual products of digestioncan be checked after the fact for actual or predicted binding to otherMHC molecules. Selected results are shown in Table 8. TABLE 8 PredictedHLA binding by proteasomally generated fragments VII. SEQ ID NO VIII.PEPTIDE HLA SYFPEITHI NIH 39 & (40) (R)MMNDQLMFL A*0201 24(23) 1360(722)A*0205 NP†  71(42)  A26 15 NP B*2705 12 50 41 RMMNDQLMF B*2705 17 75†No prediction

As seen in Table 8, N-terminal addition of authentic sequence toepitopes can generate still useful, even better epitopes, for the sameor different MHC restriction elements. Note for example the pairing of(R)MMNDQLMFL (SEQ ID NOS. 39 and (40)) with HLA-A*02, where the 10-merretains substantial predicted binding potential.

HLA-A*0201 Binding Assay

HLA-A*0201 binding studies were preformed, essentially as described inExample 1.1 above, with PSMA₆₆₃₋₆₇₁, (SEQ ID NO. 39) and PSMA₆₆₂₋₆₇₁,RMMNDQLMFL (SEQ NO. 67). As seen in FIGS. 4, 7 and 8, this epitopeexhibits significant binding at even lower concentrations than thepositive control peptide (FLPSDYFPSV (HBV₁₈₋₂₇); SEQ ID NO. 108). Thoughnot run in parallel, comparison to the controls suggests thatPSMA₆₆₂₋₆₇₁ (which approaches the Melan A peptide in affinity) has thesuperior binding activity of these two PSMA peptides.

Example 2

A multi-center clinical study is carried out using compositions asdisclosed herein. The results of the study show the compositions to beuseful and effective for debulking solid tumors and for generallyinducing anti-angiogenic activity.

Example 3 Evaluation of a PSMA composition in the Xenotransplanted HumanVasculature Model

Generation of Target Antigen-Reactive CTL

A. In Vivo Immunization of Mice.

HHD1 transgenic A*0201 mice (Pascolo, S., et al. J. Exp. Med.185:2043-2051, 1997) were anesthetized and injected subcutaneously atthe base of the tail, avoiding lateral tail veins, using 100 μlcontaining 100 nmol of PSMA₂₈₈₋₂₉₇ (SEQ ID NO. 21) and 20 μg of a HTLepitope peptide in PBS emulsified with 50 μl of IFA (incomplete Freund'sadjuvant).

B. Preparation of Stimulating Cells (LPS Blasts).

Using spleens from 2 naïve mice for each group of immunized mice,un-immunized mice were sacrificed and their carcasses placed in alcohol.Using sterile instruments, the top dermal layer of skin on the mouse'sleft side (lower mid-section) was cut through, exposing the peritoneum.The peritoneum was saturated with alcohol, and the spleen asepticallyextracted. The spleens were placed in a petri dish with serum-freemedia. Splenocytes were isolated by using sterile plungers from 3 mlsyringes to mash the spleens. Cells were collected in a 50 ml conicaltubes in serum-free media, rinsing dish well. Cells were centrifuged(12000 rpm, 7 min) and washed one time with RPMI. Fresh spleen cellswere resuspended to a concentration of 1×10⁶ cells per ml in RPMI-10%FCS (fetal calf serum). 25 g/ml lipopolysaccharide and 7 μg/ml DextranSulfate were added. Cell were incubated for 3 days in T-75 flasks at 37°C., with 5% CO₂. Splenic blasts were collected in 50 ml tubes pelleted(12,000 rpm, 7 min) and resuspended to 3×10⁷/ml in RPMI. The blasts werepulsed with the priming peptide at 50 μg/ml, RT 4 hr. mitomycinC-treated at 25 μg/ml, 37° C., 20 min and washed three times with DMEM.

C. In Vitro Stimulation.

Three days after LPS stimulation of the blast cells and the same day aspeptide loading, the primed mice were sacrificed (at 14 days postimmunization) to remove spleens as above. 3×10⁶ splenocytes wereco-cultured with 1×10⁶ LPS blasts/well in 24-well plates at 37° C., with5% CO₂ in DMEM media supplemented with 10% FCS, 5×10⁻⁵ Mβ(beta)-mercaptoethanol, 100 μg/ml streptomycin and 100 μg/mlpenicillin. Cultures were fed 5% (vol/vol) ConA supernatant on day 3 andcan be transferred on day 7. An aliquot of the CTL are also tested in astandard chromium release assay to ensure activity.

Implantation and Adoptive Transfer

1×10⁶ telomerase-transformed HDMEC in 10 μl of EGM-2-VM medium(Clonetics, San Diego, Calif.) are mixed with 0.5 ml of MATRIGEL (BectonDickinson) on ice. The mixture is injected subcutaneously, through a 25gauge needle, along the ventral midline of the thorax of SCID mice. Oneweek later 1×10⁷ T cells (target epitope-reactive or sham-immunized) in0.2 ml are injected intravenously (alternatively they can be injectedintraperitoneally).

Assessment (Micromorphometry)

At one and two weeks after transfer remove implants, fix in 10% bufferedovernight, embed in paraffin, and section. For immunofluorescencedetection of human microvessels using anti-human type IV collagen IgGand fluorescently-labeled secondary antibody, deparifinize and retrieveantigen by microwaving thin sections 2×7 minutes in 10 mM citric acid,pH 6.0. Vessel density is assessed as a function of the average numberof positively stained annular structures observed in five separate,randomly selected 20× fields-of-view, from at least three sections perimplant.

Example 4 A Fibronectin ED-B Vaccine in the HLA-transgenic Mouse Model

A. Establishment of Tumor

M1 tumor cells grown in complete RPMI plus 10% serum were harvested andwashed with PBS by centrifugation. The cells were suspended in PBS at5×10⁶ cells/ml and 0.5 ml of the suspension (early passage) was injectedsubcutaneously into the abdomen.

B. Vaccination

A nucleotide sequence encoding an HLA-A2-restricted fibronectin ED-Bdomain-derived housekeeping epitope, for example ED-B₂₉₋₃₈ (SEQ ID NO.103), is inserted into an appropriate vaccine vector (e.g. pVAX1(Invitrogen Inc, Carlsbad, Calif.) or one of the vectors described inU.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORSENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed on Apr. 28,2001, and incorporated by reference above. HHD-A2 mice are injectedintranodally in the inguinal lymph node with 0, 2, 10, 50, 100, and 200μg of vector in PBS every other day over 8 days (4 injections)alternating sides for each injection (single dosage per mouse or groupof mice). Injection series are started the day of tumor cell injection,at 2 weeks before, and at 4 and 10 weeks after.

C. Evaluation

At approximately 12 weeks after injection of tumor cells visible tumorsare observed in the mice receiving the vehicle instead of the vaccine.Effectiveness of the vaccine is expressed as the proportion ofvaccinated animals that fail to develop a tumor in the same time frame,the relative size of tumors at the same time point, the delay in timebefore tumors appear in the vaccinated animals, and the dose and numberof composition cycles needed to inhibit or prevent the establishment oftumor.

D. Alternative Schedule

The availability of more aggressive later passage M1 cells enables amore compressed experimental schedule. Instead mice are vaccinated onthe day of tumor cell inoculation, 1 and 2 weeks before, and 3 or 4 daysafter injections of 1×10⁶ cells. Effectiveness of vaccination isassessed at approximately 10 days after tumor cell inoculation.

E. Immunization with peptide

HHD-A2 mice were immunized with ED-B29-38 (SEQ ID NO. 103) in completeFreund's adjuvants and spleen cells were harvested and re-stimulated invitro using standard methodology. The resulting CTL were able tospecifically lyse peptide cells, which are HLA-A2+(FIG. 9).

Example 5 Epitopes and Epitope Clusters

Table 9 discloses epitopes and epitope clusters from PSMA and ED-B thatcan be useful in construction of compositions according to the presentinvention. TABLE 9 SEQ ID NOS.* SEQ ID NO ENTITY SEQUENCE 1 PSMA proteinAccession number**: NP_004467 2 PSMA cDNA Accession number: NM_004476 3PSMA 163-192 AFSPQGMPEGDLVYVNYARTEDFFKLERDM 4 PSMA 168-190GMPEGDLVYVNYARTEDFFKLER 5 PSMA 169-177 MPEGDLVYV 6 PSMA 168-177GMPEGDLVYV 7 PSMA 168-176 GMPEGDLVY 8 PSMA 167-176 QGMPEGDLVY 9 PSMA169-176 MPEGDLVY 10 PSMA 171-179 EGDLVYVNY 11 PSMA 170-179 PEGDLVYVNY 12PSMA 174-183 LVYVNYARTE 13 PSMA 177-185 VNYARTEDF 14 PSMA 176-185YVNYARTEDF 15 PSMA 178-186 NYARTEDFF 16 PSMA 179-186 YARTEDFF 17 PSMA181-189 RTEDFFKLE 18 PSMA 281-310 RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG 19 PSMA283-307 IAEAVGLPSIPVHPIGYYDAQKLLE 20 PSMA 289-297 LPSIPVHPI 21 PSMA288-297 GLPSIPVHPI 22 PSMA 297-305 IGYYDAQKL 23 PSMA 296-305 PIGYYDAQKL24 PSMA 291-299 SIPVHPIGY 25 PSMA 290-299 PSIPVHPIGY 26 PSMA 292-299IPVHPIGY 27 PSMA 299-307 YYDAQKLLE 28 PSMA 454-481SSIEGNYTLRVDCTPLMYSLVHLTKEL 29 PSMA 456-464 IEGNYTLRV 30 PSMA 455-464SIEGNYTLRV 31 PSMA 457-464 EGNYTLRV 32 PSMA 461-469 TLRVDCTPL 33 PSMA460-469 YTLRVDCTPL 34 PSMA 462-470 LRVDCTPLM 35 PSMA 463-471 RVDCTPLMY36 PSMA 462-471 LRVDCTPLMY 37 PSMA 653-687FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY 38 PSMA 660-681VLRMMNDQLMFLERAFIDPLGL 39 PSMA 663-671 MMNDQLMFL 40 PSMA 662-671RMMNDQLMFL 41 PSMA 662-670 RMMNDQLMF 42 PSMA 4-12 LLHETDSAV 43 PSMA13-21 ATARRPRWL 44 PSMA 53-61 TPKHNMKAF 45 PSMA 64-73 ELKAENIKKF 46 PSMA69-77 NIKKFLH¹NF 47 PSMA 68-77 ENIKKFLH¹NF 48 PSMA 220-228 AGAKGVILY 49PSMA 468-477 PLMYSLVHNL 50 PSMA 469-477 LMYSLVHNL 51 PSMA 463-471RVDCTPLMY 52 PSMA 465-473 DCTPLMYSL 53 PSMA 507-515 SGMPRISKL 54 PSMA506-515 FSGMPRISKL 55 PSMA 211-218 GNKVKNAQ 56 PSMA 202-209 IARYGKVF 57PSMA 217-225 AQLAGAKGV 58 PSMA 207-215 KVFRGNKVK 59 PSMA 211-219GNKVKNAQL 60 PSMA 269-277 TPGYPANEY 61 PSMA 268-277 LTPGYPANEY 62 PSMA271-279 GYPANEYAY 63 PSMA 270-279 PGYPANEYAY 64 PSMA 266-274 DPLTPGYPA65 PSMA 492-500 SLYESWTKK 66 PSMA 491-500 KSLYESWTKK 67 PSMA 486-494EGFEGKSLY 68 PSMA 485-494 DEGFEGKSLY 69 PSMA 498-506 TKKSPSPEF 70 PSMA497-506 WTKKSPSPEF 71 PSMA 492-501 SLYESWTKKS 72 PSMA 725-732 WGEVKRQI73 PSMA 724-732 AWGEVKRQI 74 PSMA 723-732 KAWGEVKRQI 75 PSMA 723-730KAWGEVKR 76 PSMA 722-730 SKAWGEVKR 77 PSMA 731-739 QIYVAAFTV 78 PSMA733-741 YVAAFTVQA 79 PSMA 725-733 WGEVKRQIY 80 PSMA 727-735 EVKRQIYVA 81PSMA 738-746 TVQAAAETL 82 PSMA 737-746 FTVQAAAETL 83 PSMA 729-737KRQIYVAAF 84 PSMA 721-729 PSKAWGEVK 85 PSMA 723-731 KAWGEVKRQ 86 PSMA100-108 WKEFGLDSV 87 PSMA 99-108 QWKEFGLDSV 88 PSMA 102-111 EFGLDSVELA89 ED-B domain of EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRI FibronectinTVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGID YDISVITLINGGESAPTTLTQQT 90 ED-B domainof CTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIP Fibronectin withEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRI flanking sequenceTVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGID from FribronectinYDISVITLINGGESAPTTLTQQT AVPPPTDLRFTNIGPDTMRVTW 91 ED-B domain ofAccession number: X07717 Fibronectin cds 92 ED-B 4′-5 TIIPEVPQL 93 ED-B5′-5 DTTIIPEVPQL 94 ED-B 1-10 EVPQLTDLSF 95 ED-B 23-30 TPLNSSTI 96 ED-B18-25 IGLRWTPL 97 ED-B 17-25 SIGLRWTPL 98 ED-B 25-33 LNSSTHGY 99 ED-B24-33 PLNSSTIIGY 100 ED-B 23-31 TPLNSSTII 101 ED-B 31-38 IGYRITVV 102ED-B 30-38 IIGYRITVV 103 ED-B 29-38 TIIGYRITVV 104 ED-B 31-39 IGYRITVVA105 ED-B 30-39 IIGYRITVVA 106 Melan-A 26-35_(A > L) ELAGIGILTV 107Melan-A 26-35 EAAGIGILTV 108 HBV 18-27 FLPSDYFPSV 109 HLA-B44 binderAEMGKYSFY¹This H was reported as Y in the SWISSPROT database.*Any of SEQ ID NOS. 5-17, 20-27, 29-36, 39-88, and 92-105 can be usefulas epitopes in the various embodiments of the invention. Any of SEQ IDNOS. 3, 4, 18, 19, 28, 37, 38, 89 and 90 can be useful as sequencescontaining epitopes or epitope clusters, as described in variousembodiments of the invention.**All accession numbers used here and throughout can be accessed throughthe NCBI databases, for example, through the Entrez seek and retrievalsystem on the world wide web.

PSMA LOCUS NM_004476  2653 bp  mRNA  PRI  01-NOV-2000 DEFINITION Homosapiens folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1),mRNA. ACCESSION NM_004476 VERSION NM_004476.1 GI:4758397 KEYWORDS .SOURCE human. ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata;Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Primates;Catarrhini; Hominidae; Homo. REFERENCE 1 (bases 1 to 2653) AUTHORSIsraeli, R. S., Powell, C. T., Fair, W. R. and Heston, W. D. TITLEMolecular cloning of a complementary DNA encoding a prostate-specificmembrane antigen JOURNAL Cancer Res. 53 (2), 227-230 (1993) MEDLINE93113576 REFERENCE 2 (bases 1 to 2653) AUTHORS Rinker-Schaeffer C. W.,Hawkins A. L., Su S. L., Israeli R. S., Griffin C. A., Isaacs J. T. andHeston W. D. TITLE Localization and physical mapping of theprostate-specific membrane antigen (PSM) gene to human chromosome 11JOURNAL Genomics 30 (1), 105-108 (1995) MEDLINE 96129312 PUBMED 8595888REFERENCE 3 (bases 1 to 2653) AUTHORS O'Keefe D. S., Su S. L., Bacich D.J., Horiguchi Y., Luo Y., Powell C. T., Zandvliet D., Russell P. J.,Molloy P. L., Nowak N. J., Shows T. B., Mullins C., Vonder Haar R. A.,Fair W. R. and Heston W. D. TITLE Mapping, genomic organization andpromoter analysis of the human prostate-specific membrane antigen geneJOURNAL Biochim. Biophys. Acta 1443 (1-2), 113-127 (1998) MEDLINE99057588 PUBMED 9838072 REFERENCE 4 (bases 1 to 2653) AUTHORS Maraj B.H., Leek J. P., Karayi M., Ali M., Lench N. J. and Markham A. F. TITLEDetailed genetic mapping around a putative prostate-specific membraneantigen locus on human chromosome 11p11.2 JOURNAL Cytogenet. Cell Genet.81 (1), 3-9 (1998) MEDLINE 98358137 PUBMED 9691167 COMMENT PROVISIONALREFSEQ: This record has not yet been subject to final NCBI review. Thereference sequence was derived from M99487.1. FEATURES     Location/Qualifiers source      1 . . . 2653      /organism=“Homosapiens”      /db_xref=“taxon:9606”      /chromosome=“11”     /map=“11p11.2”      /sex=“male”      /cell_line=“LNCaP-ATCC”     /cell_type=“prostate”      /tissue_type=“prostatic carcinomametastatic lymph node”      /tissue_lib=“LNCaP cDNA of Ron Israeli” gene     1 . . . 2653      /gene=“FOLH1”      /note=“FOLH; PSM; PSMA”     /db_xref=“LocusID:2346”      /db_xref=“MIM:600934” CDS      262 . .. 2514      /gene=“FOLH1”      /note=“folate hydrolase 1(prostate-specific membrane      antigen)”      /codon_start=1     /db_xref=“LocusID:2346”      /db_xref=“MIM:600934”     /evidence=experimental      /product=“folate hydrolase(prostate-specific membrane      antigen) 1”     /protein_id=“NP_004467.1”      /db_xref=“GI:4758398” (SEQ ID NO. 1)/translation=“MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA”misc feature 778 . . . 1029 /note=“PA; Region: PA domain” BASE COUNT 782a  524 c  640 g  707 t ORIGIN (SEQ ID NO. 2) 1 ctcaaaaggg gccggatttccttctcctgg aggcagatgt tgcctctctc tctcgctcgg 61 attggttcag tgcactctagaaacactgct gtggtggaga aactggaccc caggtctgga 121 gcgaattcca gcctgcagggctgataagcg aggcattagt gagattgaga gagactttac 181 cccgccgtgg tggttggagggcgcgcagta gagcagcagc acaggcgcgg gtcccgggag 241 gccggctctg ctcgcgccgagatgtggaat ctccttcacg aaaccgactc ggctgtggcc 301 accgcgcgcc gcccgcgctggctgtgcgct ggggcgctgg tgctggcggg tggcttcttt 361 ctcctcggct tcctcttcgggtggtttata aaatcctcca atgaagctac taacattact 421 ccaaagcata atatgaaagcatttttggat gaattgaaag ctgagaacat caagaagttc 481 ttatataatt ttacacagataccacattta gcaggaacag aacaaaactt tcagcttgca 541 aagcaaattc aatcccagtggaaagaattt ggcctggatt ctgttgagct agcacattat 601 gatgtcctgt tgtcctacccaaataagact catcccaact acatctcaat aattaatgaa 661 gatggaaatg agattttcaacacatcatta tttgaaccac ctcctccagg atatgaaaat 721 gtttcggata ttgtaccacctttcagtgct ttctctcctc aaggaatgcc agagggcgat 781 ctagtgtatg ttaactatgcacgaactgaa gacttcttta aattggaacg ggacatgaaa 841 atcaattgct ctgggaaaattgtaattgcc agatatggga aagttttcag aggaaataag 901 gttaaaaatg cccagctggcaggggccaaa ggagtcattc tctactccga ccctgctgac 961 tactttgctc ctggggtgaagtcctatcca gatggttgga atcttcctgg aggtggtgtc 1021 cagcgtggaa atatcctaaatctgaatggt gcaggagacc ctctcacacc aggttaccca 1081 gcaaatgaat atgcttataggcgtggaatt gcagaggctg ttggtcttcc aagtattcct 1141 gttcatccaa ttggatactatgatgcacag aagctcctag aaaaaatggg tggctcagca 1201 ccaccagata gcagctggagaggaagtctc aaagtgccct acaatgttgg acctggcttt 1261 actggaaact tttctacacaaaaagtcaag atgcacatcc actctaccaa tgaagtgaca 1321 agaatttaca atgtgataggtactctcaga ggagcagtgg aaccagacag atatgtcatt 1381 ctgggaggtc accgggactcatgggtgttt ggtggtattg accctcagag tggagcagct 1441 gttgttcatg aaattgtgaggagctttgga acactgaaaa aggaagggtg gagacctaga 1501 agaacaattt tgtttgcaagctgggatgca gaagaatttg gtcttcttgg ttctactgag 1561 tgggcagagg agaattcaagactccttcaa gagcgtggcg tggcttatat taatgctgac 1621 tcatctatag aaggaaactacactctgaga gttgattgta caccgctgat gtacagcttg 1681 gtacacaacc taacaaaagagctgaaaagc cctgatgaag gctttgaagg caaatctctt 1741 tatgaaagtt ggactaaaaaaagtccttcc ccagagttca gtggcatgcc caggataagc 1801 aaattgggat ctggaaatgattttgaggtg ttcttccaac gacttggaat tgcttcaggc 1861 agagcacggt atactaaaaattgggaaaca aacaaattca gcggctatcc actgtatcac 1921 agtgtctatg aaacatatgagttggtggaa aagttttatg atccaatgtt taaatatcac 1981 ctcactgtgg cccaggttcgaggagggatg gtgtttgagc tagccaattc catagtgctc 2041 ccttttgatt gtcgagattatgctgtagtt ttaagaaagt atgctgacaa aatctacagt 2101 atttctatga aacatccacaggaaatgaag acatacagtg tatcatttga ttcacttttt 2161 tctgcagtaa agaattttacagaaattgct tccaagttca gtgagagact ccaggacttt 2221 gacaaaagca acccaatagtattaagaatg atgaatgatc aactcatgtt tctggaaaga 2281 gcatttattg atccattagggttaccagac aggccttttt ataggcatgt catctatgct 2341 ccaagcagcc acaacaagtatgcaggggag tcattcccag gaatttatga tgctctgttt 2401 gatattgaaa gcaaagtggacccttccaag gcctggggag aagtgaagag acagatttat 2461 gttgcagcct tcacagtgcaggcagctgca gagactttga gtgaagtagc ctaagaggat 2521 tctttagaga atccgtattgaatttgtgtg gtatgtcact cagaaagaat cgtaatgggt 2581 atattgataa attttaaaattggtatattt gaaataaagt tgaatattat atataaaaaa 2641 aaaaaaaaaa aaa

ED-B domain of Fibronectin LOCUS HSFIBEDB  2823 bp  DNA  linear PRI09-AUG-1999 DEFINITION Human fibronectin gene ED-B region. ACCESSIONX07717 VERSION X07717.1 GI:31406 KEYWORDS alternate splicing;fibronectin. SOURCE human. ORGANISM Homo sapiens. Eukaryota; Metazoa;Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria;Primates; Catarrhini; Hominidae; Homo. REFERENCE 1 (bases 1 to 2823)AUTHORS Paolella, G., Henchcliffe, C., Sebastio, G. and Baralle, F. E.TITLE Sequence analysis and in vivo expression show that alternativesplicing of ED-B and ED-A regions of the human fibronectin gene areindependent events JOURNAL Nucleic Acids Res. 16 (8), 3545-3557 (1988)MEDLINE 88233940 FEATURES Location/Qualifiers source 1 . . . 2823/organism=“Homo sapiens” /db_xref=“taxon:9606” /clone=“MA10” exon 1 . .. 104 /number=1 /product=“fibronectin” CDS join(<2 . . . 104, 1375 . . .1647, 2758 . . . >2823) /codon_start=1 /product=“fibronectin”/protein_id=“CAB52437.1” /db_xref=∫GI:5725425” (SEQ ID NO. 90)/translation=“CTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTMRVTW” intron 105 . . . 1374 /number=1 exon 1375 . . . 1647 /note=“ED-Bexon” /number=2 /product=“fibronectin” intron 1648 . . . 2757 /number=2exon 2758 . . . 2823 /number=3 /product=“fibronectin” BASE COUNT 824a  556 c  528 g  91 t ORIGIN (SEQ ID NO. 91) 1 ctgcactttt gataacctgagtcccggcct ggagtacaat gtcagtgttt acactgtcaa 61 ggatgacaag gaaagtgtccctatctctga taccatcatc ccaggtaata gaaaataagc 121 tgctatcctg agagtgacattccaataaga gtggggatta gcatcttaat ccccagatgc 181 ttaagggtgt caactatatttgggatttaa ttccgatctc ccagctgcac tttccaaaac 241 caagaagtca aagcagcgatttggacaaaa tgcttgctgt taacactgct ttactgtctg 301 tgcttcactg ggatgctgtgtgttgcagcg agtatgtaat ggagtggcag ccatggcttt 361 aactctgtat tgtctgctcacatggaagta tgactaaaac actgtcacgt gtctgtactc 421 agtactgata ggctcaaagtaatatggtaa atgcatccca tcagtacatt tctgcccgat 481 tttacaatcc atatcaatttccaacagctg cctatttcat cttgcagttt caaatccttc 541 tttttgaaaa ttggattttaaaaaaaagtt aagtaaaagt cacaccttca gggttgttct 601 ttcttgtggc cttgaaagacaacattgcaa aggcctgtcc taaggatagg cttgtttgtc 661 cattgggtta taacataatgaaagcattgg acagatcgtg tccccctttg gactcttcag 721 tagaatgctt ttactaacgctaattacatg ttttgattat gaatgaacct aaaatagtgg 781 caatggcctt aacctaggcctgtctttcct cagcctgaat gtgcttttga atggcacatt 841 tcacaccata cattcataatgcattagcgt tatggccatg atgttgtcat gagttttgta 901 tgggagaaaa aaaatcaatttatcacccat ttattatttt ttccggttgt tcatgcaagc 961 ttattttcta ctaaaacagttttggaatta ttaaaagcat tgctgatact tacttcagat 1021 attatgtcta ggctctaagaatggtttcga catcctaaac agccatatga tttttaggaa 1081 tctgaacagt tcaaattgtaccctttaagg atgttttcaa aatgtaaaaa atatatatat 1141 atatatatat tccctaaaagaatattcctg tttattcttc tagggaagca aactgttcat 1201 gatgcttagg aagtcttttcagagaattta aaacagattg catattacca tcattgcttt 1261 aacattccac caattttactactagtaacc tgatatacac tgctttattt tttcctcttt 1321 ttttccctct attttccttttgcctccccc tccctttgct ttgtaactca atagaggtgc 1381 cccaactcac tgacctaagctttgttgata taaccgattc aagcatcggc ctgaggtgga 1441 ccccgctaaa ctcttccaccattattgggt accgcatcac agtagttgcg gcaggagaag 1501 gtatccctat ttttgaagattttgtggact cctcagtagg atactacaca gtcacagggc 1561 tggagccggg cattgactatgatatcagcg ttatcactct cattaatggc ggcgagagtg 1621 cccctactac actgacacaacaaacgggtg aattttgaaa acttctgcgt ttgagacata 1681 gatggtgttg catgctgccaccagttactc cggttaaata tggatgtttc atgggggaag 1741 tcagcaattg gccaaagattcagataggtg gaattggggg gataaggaat caaatgcatc 1801 tgctaaactg attggagaaaaacacatgca atatcttcag tacactctca tttaaaccac 1861 aagtagatat aaagcctagagaaatacaga tgtctgctct gttaaatata aaatagcaaa 1921 tgttcattca atttgaagacctagaatttt tcttcttaaa taccaaacac gaataccaaa 1981 ttgcgtaagt accaattgataagaatatat caccaaaatg taccatcatg ctcttccttc. 2041 taccctttga taaactctaccatgctcctt ctttgtagct aaaaacccat caaaatttag 2101 ggtagagtgg atgggcattgttttgaggta ggagaaaagt aaacttggga ccattctagg 2161 ttttgttgct gtcactaggtaaagaaacac ctctttaacc acagtctggg gacaagcatg 2221 caacattfta aaggttctctgctgtgcatg ggaaaagaaa catgctgaga accaatttgc 2281 atgaacatgt tcacttgtaagtagaattca ctgaatggaa ctgtagctct agatatctca 2341 catgggggga agtttaggaccctcttgtct ttttgtctgt gtgcatgtat ttctttgtaa 2401 agtactgcta tgtttctctttgctgtgtgg caacttaagc ctcttcggcc tgggataaaa 2461 taatctgcag tggtattaataatgtacata aagtcaacat atttgaaagt agattaaaat 2521 cttttttaaa tatatcaatgatggcaaaaa ggttaaaggg ggcctaacag tactgtgtgt 2581 agtgttttat ttttaacagtagtacactat aacttaaaat agacttagat tagactgttt 2641 gcatgattat gattctgtttcctttatgca tgaaatattg attttacctt tccagctact 2701 tcgttagctt taattttaaaatacattaac tgagtcttcc ttcttgttcg aaaccagctg 2761 ttcctcctcc cactgacctgcgattcacca acattggtcc agacaccatg cgtgtcacct 2821 ggg //

1. A method of treating neoplastic disease comprising the step ofimmunizing a mammal to induce a cellular immune response directedagainst a first antigen differentially expressed by tumor-associatedneovasculature and a second antigen associated with a tumor, wherein theimmunization comprises delivering at least a first immunogencorresponding to the first antigen and a second immunogen correspondingto the second antigen.
 2. The method of claim 1, wherein the cellularimmune response comprises a CTL response.
 3. The method of claim 1further comprising the step of detecting the cellular immune response.4. The method of claim 3, wherein the detecting step comprises detectionof tumor growth inhibition, tumor size reduction, inhibition of tumormetastasis, or increase in life expectancy of the mammal.
 5. The methodof claim 3, wherein the detecting step comprises an assay selected fromthe group consisting of a cytokine assay, a chromium release assay, animmunofluorescence assay, a cytotoxic T lymphocyte (CTL) assay, anElispot assay, and observation of the health of the mammal.
 6. A methodof treating neoplastic disease comprising the step of immunizing amammal to induce a cellular immune response directed against an antigendifferentially expressed by tumor-associated neovasculature, wherein theimmunization comprises delivering a first immunogen comprising at leastone housekeeping epitope and a second immunogen comprising at least oneimmune epitope, wherein the housekeeping and immune epitopes are derivedfrom said antigen differentially expressed by tumor-associatedneovasculature.
 7. The method of claim 6, wherein the first immunogenand the second immunogen are the same.
 8. The method of claim 6, furthercomprising the step of treating the mammal with an anti-tumor therapyactive directly against cancerous cells.
 9. The method of claim 8,wherein the anti-tumor therapy comprises immunization against atumor-associated antigen.
 10. The method of claim 6, wherein thecellular immune response comprises a CTL response.
 11. The method ofclaim 6, further comprising the step of detecting the cellular immuneresponse.
 12. The method of claim 11, wherein the detecting stepcomprises detection of tumor growth inhibition, tumor size reduction,inhibition of tumor metastasis, or increase in life expectancy of themammal.
 13. The method of claim 11, wherein the detecting step comprisesan assay selected from the group consisting of a cytokine assay, achromium release assay, an immunofluorescence assay, a cytotoxic Tlymphocyte (CTL) assay, an Elispot assay, and observation of the healthof the mammal.
 14. A method of treating neoplastic disease comprisingthe step of immunizing a mammal to induce a cellular immune responsedirected against an antigen differentially expressed by tumor-associatedneovasculature, wherein the immunization comprises delivering animmunogen comprising at least one housekeeping epitope derived from saidantigen differentially expressed by tumor-associated neovasculature. 15.The method of claim 14, wherein the cellular immune response comprises aCTL response.
 16. The method of claim 14, further comprising the step ofdetecting the cellular immune response.
 17. The method of claim 16,wherein the detecting step comprises detection of tumor growthinhibition, tumor size reduction, inhibition of tumor metastasis, orincrease in life expectancy of the mammal.
 18. The method of claim 16,wherein the detecting step comprises an assay selected from the groupconsisting of a cytokine assay, a chromium release assay, animmunofluorescence assay, a cytotoxic T lymphocyte (CTL) assay, anElispot assay, and observation of the health of the mammal.